EP0677055A1 - Antisense molecules directed against a fibroflast growth factor receptor gene family - Google Patents

Antisense molecules directed against a fibroflast growth factor receptor gene family

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Publication number
EP0677055A1
EP0677055A1 EP94907109A EP94907109A EP0677055A1 EP 0677055 A1 EP0677055 A1 EP 0677055A1 EP 94907109 A EP94907109 A EP 94907109A EP 94907109 A EP94907109 A EP 94907109A EP 0677055 A1 EP0677055 A1 EP 0677055A1
Authority
EP
European Patent Office
Prior art keywords
polynucleotide
seq
gene
growth factor
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP94907109A
Other languages
German (de)
French (fr)
Inventor
Larry A. Denner
Ajay A. Rege
Richard A. F. Dixon
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Encysive Pharmaceuticals Inc
Original Assignee
Texas Biotechnology Corp
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Filing date
Publication date
Application filed by Texas Biotechnology Corp filed Critical Texas Biotechnology Corp
Publication of EP0677055A1 publication Critical patent/EP0677055A1/en
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates

Definitions

  • the present invention relates to growth-factor related polynucleotides and their use in inhibiting the proliferation of smooth muscle cells, and more specifically to antisense molecules corresponding in sequence to portions of a gene for fibroblast growth factor receptor, and their use in inhibiting the proliferation of smooth muscle cells.
  • Antisense polynucleotides contain artificial sequences of nucleotide bases complementary to messenger RNA (mRNA or message) or the sense strand of double stranded DNA. Admixture of sense and antisense oligo- or polynucleotides under appropriate conditions leads to binding of the two molecules, or hybridization.
  • FGF fibroblast growth factor
  • Fibroblast growth factors mediate their cellular responses through binding and activation of high affinity cell surface receptors known as fibroblast growth factor receptors. To date, five such receptors have been identified and cloned from humans, and all appear to contain tyrosine kinase activity. Jaye et al., Bioch. Biophy ⁇ . Acta 1135:185-199 (1992).
  • Activated smooth muscle cells elaborate growth factors such as platelet derived growth factor (PDGF) , basic and acidic fibroblast growth factor, interleukins and transforming growth factor ⁇ .
  • PDGF platelet derived growth factor
  • FGF receptor FGF receptor
  • epidermal growth factor receptor epidermal growth factor receptor.
  • SMC Activation of SMC, leading to the proliferation of those cells, occurs in response to a number of stimuli, including surgical procedures such as coronary angioplasty.
  • the proliferation of SMC results in such disease states as atherosclerosis and restenosis.
  • This invention is applicable to a number of disease states in which the proliferation of smooth muscle cells is involved, including, but not limited to, vascular stenosis, post-angioplasty restenosis (including coronary, carotid and peripheral stenosis) , other non-angioplasty reopening procedures such as atherectomy and laser procedures, atherosclerosis, atrial-venous shunt failure, cardiac hypertrophy, vascular surgery, and organ transplant.
  • vascular stenosis including coronary, carotid and peripheral stenosis
  • other non-angioplasty reopening procedures such as atherectomy and laser procedures, atherosclerosis, atrial-venous shunt failure, cardiac hypertrophy, vascular surgery, and organ transplant.
  • the present invention is directed to a polynucleotide of about 10 to about 50, preferably about 15 to about 25, and more preferably about 20, nucleic acid bases in length, which polynucleotide hybridizes to the gene encoding fibroblast growth factor receptor.
  • a preferred polynucleotide is an antisense molecule having the sequence shown in SEQ ID NO:l.
  • GCGCCCCCAG CTGACCAT (SEQ ID NO:2), GCAGGCCGGG ACTACCAT (SEQ ID NO:3), GGCCAAGAGC AGCCACAT (SEQ ID NO:4), GCACTTCCAG CTCCACAT (SEQ ID NO:5), ACGACCCCAG CTGACCAT (SEQ ID NO:6), GCAGGCAGGG GCGCCCAT (SEQ ID NO:7), GGCCAGCAGC AGCCGCAT (SEQ ID NO:8),
  • GCAGGCCGGG ACTACCAT SEQ ID NO:9
  • GGGGATCCTC AGGGAGCT SEQ ID NO:27
  • CAGTTTCTTC TCCATTTT SEQ ID NO:28
  • the bases of the polynucleotide molecule are linked by pseudophosphate bonds that are resistant to cleavage by exonuclease and/or endonuclease enzymes.
  • Preferred pseudophosphate bonds are phosphorothioate bonds.
  • the present invention is further directed to a polynucleotide of about 10 to about 50, preferably about 20 to about 40, and more preferably about 20, nucleic acid bases, which polynucleotide hybridizes to the about 5 to about 25, preferably about 10 to about 20, and more preferably about 10, nucleic acid bases flanking the start codon of the mRNA for fibroblast growth factor receptor.
  • a preferred such polynucleotide is an antisense molecule having the sequence shown in SEQ ID NOs 1 through 9 above, as well as SEQ ID NO:10:
  • GCACTTCCAG CCCCACATCC C (SEQ ID NO:10).
  • Further preferred polynucleotides include: CTTCCAGCCC CACATCCC (SEQ ID NO:11), CCAGCCCCAC ATCCC (SEQ ID NO:12),
  • CTTCCAGCCC CACATCCCAG T (SEQ ID NO:13), GCACTTCCAG CCCCA (SEQ ID NO:14), CTTCCAGCCC CACAT (SEQ ID NO:15), GCCCCACATC CCAGTTCT (SEQ ID NO:16), CCAGCCCCAC ATCCCAGT (SEQ ID NO:17), GCCCCACATC CCAGT (SEQ ID NO:18), CTTCCAGCTC CACATCCCAG T (SEQ ID NO:19), CCAGCTCCAC ATCCCAGT (SEQ ID NO:20), GCTCCACATC CCAGT (SEQ ID NO:21), GAGGAGGCAC TTCCAGCTCC ACATCCC (SEQ ID NO:22),
  • the present invention is also directed to a pharmaceutical composition
  • a pharmaceutical composition comprising a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor, dissolved or dispersed in a physiologically tolerable diluent.
  • the present invention is further directed to a process for inhibiting vascular smooth muscle cell proliferation that comprises contacting vascular smooth muscle cells whose growth is to be inhibited in an aqueous medium suitable for growth of those cells with an inhibition-effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor and maintaining said contact in said aqueous medium under biological culture conditions for a time period sufficient for the growth of the contacted cells to be inhibited.
  • the present invention is still further directed to a process for treating vascular smooth muscle cell proliferation that comprises administering to a host mammal in need of such treatment an effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor.
  • the present invention is yet further directed to a process for treating a disease state involving the proliferation of vascular smooth muscle cells that comprises administering to a host mammal in need of such treatment an effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor.
  • Figure 1 shows the percentage of growth inhibition of smooth muscle cells upon the addition of various concentrations of either antisense polynucleotides directed against the gene for mouse fibroblast growth factor receptor 1, or antisense polynucleotides directed against the gene for plasminogen activator inhibitor 1.
  • Figure 2 shows the percentage of growth inhibition of smooth muscle cells upon the addition of either 10 ⁇ M or 50 ⁇ M of antisense polynucleotides directed against the gene for mouse fibroblast growth factor 2, 3 or 4, and the gene for plasminogen activator inhibitor 1.
  • Figure 3 shows the percentage of growth inhibition of rat carotid artery smooth muscle cells proliferation upon the addition of either 10 ⁇ M or 50 ⁇ M of antisense polynucleotides directed against mouse fibroblast growth factor gene 1, and the vinculin gene.
  • Figure 4 shows the percentage of maximal inti al thickening of rat carotid arteries after angioplasty and treatment in vivo, upon the addition of antisense polynucleotides directed against FGF receptor 1 or PAI-1, or control treatment with carrier alone.
  • the present invention is directed to a polynucleotide of about 10 to about 50 nucleic acid bases in length, which polynucleotide hybridizes to the gene for fibroblast growth factor receptor.
  • the gene for fibroblast growth factor receptor can be derived from any mammal, including mouse and humans.
  • the gene for fibroblast growth factor receptor is that of human fibroblast growth factor receptor.
  • the polynucleotide may preferably be from about 15 to about 25 nucleic acid bases in length, and more preferably about 20 nucleic acid bases in length.
  • the present invention is further directed to a polynucleotide of about 10 to about 50, preferably about 20 to about 40, and more preferably about 20, nucleic acid bases, which polynucleotide hybridizes to the about 5 to about 25, preferably about 10 to about 20, and more preferably about 10, nucleic acid bases flanking the start codon of the mRNA for fibroblast growth factor receptor.
  • the present invention contemplates a polynucleotide that hybridizes to any of the genes encoding a member of the fibroblast growth factor receptor family. Any such polynucleotide capable of inhibiting the proliferation of smooth muscle cell proliferation can be used.
  • polynucleotide refers to a covalently linked sequence of nucleotides in which the 3 # position of the pentose of one nucleotide is joined by a phosphodiester group to the 5' position of the pentose of the next nucleotide.
  • the nucleotides may be composed of deoxyribonucleotides or ribonucleotides.
  • Polynucleotide hybridization of greater than about 90 percent homology (identity) , and more preferably about 99 percent homology, is contemplated in the present invention.
  • the Polynucleotides A preferred polynucleotide is an antisense molecule having the sequence shown in SEQ ID N0:1, directed against the gene for mouse fibroblast growth factor (FGF) receptor 1.
  • FGF mouse fibroblast growth factor
  • GCGCCCCCAG CTGACCAT (SEQ ID NO: 2 ] , directed against the gene for mouse FGF receptor 2,
  • GCAGGCCGGG ACTACCAT (SEQ ID NO:3] , directed against the gene for mouse FGF receptor 3, GGCCAAGAGC AGCCACAT (SEQ ID NO: ] , directed against the gene for mouse FGF receptor 4,
  • GCACTTCCAG CTCCACAT directed against the gene for human FGF receptor 1
  • ACGACCCCAG CTGACCAT directed against the gene for human FGF receptor 2
  • GCAGGCAGGG GCGCCCAT (SEQ ID NO: 1 ) , directed against the gene for human FGF receptor 3,
  • GGCCAGCAGC AGCCGCAT directed against the gene for human FGF receptor 4
  • GCAGGCCGGG ACTACCAT directed against the gene for human FGF receptor 5.
  • sequence of the polynucleotide directed against the gene for human FGF receptor 5 shown in SEQ ID NO:9 is identical to the polynucleotide directed against the gene for mouse FGF receptor 3 shown in SEQ ID NO:3; however, different SEQ ID NOs will be used to avoid confusion and because of the fact that the human and mouse sequences differ in other locations.
  • polynucleotides include GGGGATCCTC AGGGAGCT (SEQ ID NO:27) , and CAGTTTCTTC TCCATTTT (SEQ ID NO:28), both of which are directed against internal sequences of the mouse FGF receptor 1 gene,
  • GCACTTCCAG CCCCACATCC C (SEQ ID NO:10), directed against position -3 to +18, relative to the start codon, of mouse FGF receptor gene 1,
  • CTTCCAGCCC CACATCCC (SEQ ID NO:11), directed against position -3 to +15, relative to the start codon, of mouse FGF receptor gene 1
  • CCAGCCCCAC ATCCC (SEQ ID NO:12), directed against position -3 to +12, relative to the start codon, of mouse FGF receptor gene 1
  • CTTCCAGCCC CACATCCCAG T (SEQ ID NO:13), directed against position -6 to +15, relative to the start codon, of mouse FGF receptor gene 1,
  • GCACTTCCAG CCCCA (SEQ ID NO:14), directed against position +4 to +18, relative to the start codon, of mouse FGF receptor gene 1,
  • CTTCCAGCCC CACAT (SEQ ID NO:15), directed against position +1 to +15, relative to the start codon, of mouse FGF receptor gene 1,
  • GCCCCACATC CCAGTTCT (SEQ ID NO:16), directed against position -9 to +9, relative to the start codon, of mouse FGF receptor gene 1
  • CCAGCCCCAC ATCCCAGT (SEQ ID NO:17), directed against position -6 to +12, relative to the start codon, of mouse FGF receptor gene 1
  • GCCCCACATC CCAGT (SEQ ID NO:18), directed against position -6 to +9, relative to the start codon, of mouse FGF receptor gene 1,
  • CTTCCAGCTC CACATCCCAG T (SEQ ID NO:19), directed against position -6 to +15, relative to the start codon, of human FGF receptor gene 1
  • CCAGCTCCAC ATCCCAGT (SEQ ID NO:20), directed against position -6 to +12, relative to the start codon, of human FGF receptor gene 1
  • GCTCCACATC CCAGT (SEQ ID NO:21) , directed against position -6 to +9, relative to the start codon, of human FGF receptor gene 1
  • GAGGAGGCAC TTCCAGCTCC ACATCCC (SEQ ID NO:22), directed against position -3 to +24, relative to the start codon of human FGF receptor gene 1
  • GAGGCACTTC CAGCTCCACA TCCC (SEQ ID NO:23), directed against position -3 to +21, relative to the start codon of human FGF receptor gene 1
  • GCACTTCCAG CTCCACATCC CAGT (SEQ ID NO:24), directed against position -6 to +18, relative to the start codon of human FGF receptor gene 1,
  • GCACTTCCAG CTCCACATCC C (SEQ ID NO:25) , directed against position -3 to +18, relative to the start codon of human FGF receptor gene 1, and
  • CTTCCAGCTC CACATCCC (SEQ ID NO:26) , directed against position -6 to +9, relative to the start codon of human FGF receptor gene 1.
  • the bases of the polynucleotide are linked by pseudophosphate bonds that are resistant to cleavage by exonuclease or endonuclease enzymes.
  • Exonuclease enzymes hydrolyze the terminal phosphodiester bond of a nucleic acid.
  • Endonuclease enzymes hydrolyze internal phosphodiester bonds of a nucleic acid.
  • pseudophosphate bonds include, but are not limited to, methylphosphonate, phosphomorpholidate, phosphorothioate, phosphorodithioate and phosphoroselenoate bonds.
  • exonuclease and/or endonuclease resistant polynucleotides can be obtained by blocking the 3 ' and/or 5' terminal nucleotides with substituent groups such as acridine, cholesterol or a methyl group.
  • Preferred pseudophosphate bonds are phosphorothioate bonds.
  • the pseudophosphate bonds may comprise the bonds at the 3' and or 5' terminus, the bonds from about 1 to about 5 of the 3' and/or 5' terminus bases, or the bonds of the entire polynucleotide.
  • a preferred polynucleotide with pseudophosphate bonds is one in which all of the bonds are comprised of pseudophosphate bonds.
  • DNA or RNA polynucleotides can be prepared using several different methods, as is well known in the art. See, e.g., Ausubel et al. (eds.). Current Protocols in Molecular Biology, John Wiley & Sons, New York (1990) .
  • the phosphoramidate synthesis method is described in Caruthers et al., Meth. Enzy ol. 154:287 (1987) ; the phosphorothioate polynucleotide synthesis method is described in Iyer et al., J. Am. Chem. Soc. 112:1253 (1990).
  • the present invention is also directed to a pharmaceutical composition
  • a pharmaceutical composition comprising a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor, dissolved or dispersed in a physiologically tolerable diluent.
  • the polynucleotide is from about 15 to about 25 nucleic acid bases in length, and more preferably about 20 nucleic acid bases in length.
  • the present invention includes one or more polynucleotides as described above formulated into compositions together with one or more non-toxic physiologically tolerable or acceptable diluents.
  • carriers, adjuvants or vehicles that are collectively referred to herein as diluents for parenteral injection, for oral administration in solid or liquid form, for rectal or topical administration, or the like.
  • the compositions can be administered to humans and animals either orally, rectally, parenterally (intravenous, by intramuscularly or subcutaneously) , intracisternally, intravaginally, intraperitoneally, locally (powders, ointments or drops) , or as a buccal or nasal spray.
  • compositions can also be delivered through a catheter for local delivery at the site of vascular damage, via an intracoronary catheter or stent (a tubular device composed of a fine wire mesh) , or via a biodegradable polymer.
  • the compositions may also be complexed to ligands, such as antibodies, for targeted delivery of the compositions to the site of smooth muscle cell proliferation.
  • compositions are preferably administered via parenteral delivery at the local site of smooth muscle cell proliferation.
  • the parenteral delivery is preferably via catheter.
  • compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like) , suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
  • compositions can also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents.
  • adjuvants such as preserving, wetting, emulsifying, and dispensing agents.
  • Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride and the like.
  • Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
  • Suspensions in addition to the active compounds, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
  • Dosage forms for topical administration of a compound of this invention include ointments, powders, sprays and inhalants.
  • the active component is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers or propellants as may be required. Ophthalmic formulations, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.
  • compositions of the present invention may be varied so as to obtain an amount of active ingredient that is effective to obtain a desired therapeutic response for a particular composition and method of administration.
  • the selected dosage level therefore depends upon the desired therapeutic effect, on the route of administration, on the desired duration of treatment and other factors.
  • the total daily dose of the compounds of this invention administered to a host in single or divided doses may be in amounts, for example, of from about 1 nanomol to about 5 micromols per kilogram of body weight.
  • Dosage unit compositions may contain such amounts of such submultiples thereof as may be used to make up the daily dose. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the body weight, general health, sex, diet, time and route of administration, rates of absorption and excretion, combination with other drugs and the severity of the particular disease being treated.
  • the present invention is further directed to a process for inhibiting vascular smooth muscle cell proliferation that comprises contacting vascular smooth muscle cells whose growth is to be inhibited in an aqueous medium suitable for growth of those cells with an inhibition-effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor and maintaining said contact in said aqueous medium under biological culture conditions for a time period sufficient for the growth of the contacted cells to be inhibited.
  • an “inhibition-effective amount” is that amount of a polynucleotide of the present invention which is sufficient for inhibiting the growth or killing a cell contacted with such a polynucleotide.
  • Means for determining an inhibition- effective amount in a particular subject will depend, as is well known in the art, on the nature of the polynucleotide used, the mass of the subject being treated, whether killing or growth inhibition of the cells is desired, and the like.
  • Contact is achieved by admixing the composition with a preparation of vascular smooth muscle cells.
  • Biological culture conditions are those conditions necessary to maintain the growth and replication of the vascular smooth muscle cells in a normal, polynucleotide-free environment. These biological culture conditions, encompassing such factors as temperature, humidity, atmosphere, pH and the like, must be suitable for the proliferation of vascular smooth muscle cells in the absence of polynucleotides so that the effects of such polynucleotides on relevant growth parameters can be measured.
  • a preferred polynucleotide useful in this process has the sequence shown in SEQ ID N0:1.
  • a further preferred polynucleotide useful in this process links the bases of SEQ ID NO:l by pseudophosphate bonds that are resistant to cleavage by exonuclease enzymes.
  • Preferred pseudophosphate bonds are phosphorothioate bonds.
  • polynucleotides have the sequences shown in SEQ ID NO:2 through 28.
  • the present invention is still further directed to a process for treating vascular smooth muscle cell proliferation that comprises administering to a host mammal in need of such treatment an effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor.
  • a host mammal in need of the treatment of a process for the inhibition of vascular smooth muscle cell proliferation suffers from a disease state in which such proliferation is implicated.
  • disease states include vascular stenosis, post-angioplasty restenosis (including coronary, carotid and peripheral stenosis) , other non-angioplasty reopening procedures such as atherectomy and laser procedures, atherosclerosis, atrial-venous shunt failure, cardiac hypertrophy, vascular surgery, by-pass surgery and organ transplant.
  • the polynucleotide as described above is dissolved or dispersed in a physiologically tolerable diluent.
  • the present invention is yet further directed to a process for treating a disease state involving the proliferation of vascular smooth muscle cells that comprises administering to a host mammal in need of such treatment an effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor.
  • a disease state involving the proliferation of vascular smooth muscle cells include, but not limited to, vascular stenosis, post-angioplasty restenosis (including coronary, carotid and peripheral stenosis) , other non-angioplasty reopening procedures such as atherectomy and laser procedures, atherosclerosis, atrial-venous shunt failure, cardiac hypertrophy, vascular surgery, and organ transplant.
  • vascular smooth muscle cells include, but not limited to, vascular stenosis, post-angioplasty restenosis (including coronary, carotid and peripheral stenosis) , other non-angioplasty reopening procedures such as atherectomy and laser procedures, atherosclerosis, atrial-venous shunt failure, cardiac hypertrophy, vascular surgery, and organ transplant.
  • Cleavage was facilitated by mixing of the solution every 30 minutes with the help of two 5 ml slip-tip syringes.
  • the solution was collected in a screw-capped glass vial and deprotection was accomplished either at room temperature for 24 hours or at 55 C for 5 hours.
  • the contents were transferred to a 13x100 mm glass tube, chilled on ice and evaporated to dryness using a Savant Speed-Vac.
  • the polynucleotide was then dissolved in 1 ml of 0.1M triethylammonium acetate (TEAA) , pH 7.0.
  • TEAA triethylammonium acetate
  • the oligo was detritylated and purified on a Rainin Dyna ax C18 semipreparative column (10mm x 25cm, 5um, 300 A) .
  • the mobile phases were (A): 0.1M TEAA, pH7.0, 5% acetonitrile; (B) : 95% acetonitrile, 5% water; (C) : 0.5% TFA in water.
  • the column was developed at 2ml/min with the following gradient: 10% B in A, 10 min; 100% A, 4 min; 100% C, 8 min; 100% A, 8 min; 100% A to 45% B in 24 min.
  • This procedure first separates the trityl-on full length polynucleotide from its failure sequences containing free hydroxyl groups and synthesis reagents. This is followed by the removal of 5'-DMT by 0.5% TFA. Finally the gradient resolved the desired detritylated sequence from other contaminants.
  • the polynucleotide concentration was determined by measuring the absorbance at 260 nm. Typical yields were 30-40%.
  • the integrity of the polynucleotide was determined by polyacrylamide gel electrophoresis (PAGE; 20% polyacrylamide, 7M urea) and staining with 0.2% methylene blue.
  • the pellet was resuspended in 4 ml/carotid of the following media: 20% fetal bovine serum albumin (Hyclone; FBS) ; 2mM glutamine (Gibco) ; 100 units/ml penicillin G sodium (Gibco) ; 100 ug/ml streptomycin sulfate (Gibco) ; DMEM (Gibco) .
  • FBS fetal bovine serum albumin
  • Gibco 2mM glutamine
  • penicillin G sodium Gibco
  • streptomycin sulfate Gibco
  • DMEM DMEM
  • PBS phosphate buffered saline
  • trypsin-EDTA Gibco; 0.25% trypsin-EDTA
  • the flask was rinsed with an additional 4 ml media (DMEM, 20% PBS, 2 mM glutamine, 50 units/ml penicillin, 50 ug/ml streptomycin) .
  • the trypsinized cells and the rinse were combined and centrifuged at 400 x g for 10 min.
  • the supernatant was removed and 5 is of fresh media was added to the pellet.
  • the pellet was resuspended by vigorous trituration, and the number of cells was determined using a Coulter counter.
  • the cells were diluted to 3,500 cells/100 ul and, using a 12 channel digital micropipette, '100 ul/well of the cells were seeded in a 96 well (Falcon) flat-bottom, microtiter cell culture plate. The culture plate was then incubated at 37*C in 5% C0 2 .
  • each well was rinsed twice with 100 ul PBS, and overlaid with 100 ul/well growth arrest media: 0.1% FBS (heat inactivated at 65 # C for 45 min.); 2mM glutamine; 50 units/ml penicillin; 50 ug/ml streptomycin.
  • the growth arrest media was removed.
  • the cell number was determined (treatment day counts) using a Coulter counter by averaging the cell number from three wells. To the remaining wells was added 100 ul complete media (DMEM, 10% FBS/65'C inactivated, glutamine, pen/strep) without or with antisense polynucleotides and the plates were placed in an incubator at 37*C in 5% C0 2 .
  • Cutdowns were performed on the carotid and iliac arteries and 25% bupiviamine used as a topical anesthetic.
  • a 2F embolectomy catheter was inserted into the left iliac artery and advanced to the distal end of the left carotid artery. The balloon was inflated and pulled down the artery 3 times. The catheter was then removed.
  • Antisense polynucleotide or vehicle was then delivered at 6 ⁇ l/min for 5 min with the catheter tied to the proximal portion of the artery to prevent blood from flowing around the catheter tip and washing the drug out of the artery.
  • the carotid was then ligated distal to the heart near the bifurcation of the internal and external branches of the artery. After 15 min of static incubation, the ligatures and the catheter were removed to restore normal blood flow.
  • Rats were then closed with 4.0 silk suture. Rats recovered under an infrared heating lamp connected to a proportional regulated heater. Topical antibiotics were applied to the incisions.
  • the treated section of the left carotid artery was removed and the central portion embedded in paraffin, cut in 5 ⁇ m sections, and stained with hematoxylin and eosin.
  • a frame grabber (Targa+) and image analysis software (Java) were used to measure cross-sectional areas of the medial and intimal layers. To standardize the intimal area for arteries of different size, the intimal area was divided by the medial area and this ratio used as the parameter to compare drug treatment with control.
  • Carotid arteries were dissected from male Sprague-Dawley rats weighing 200 to 300 grams.
  • DMEM Dulbecco's minimal essential medium
  • the medium was changed every three to four days.
  • trypsin was added to the growth culture to isolate cells which had grown out of the arterial explant. These isolated cells were plated in 96 well trays at a concentration of 2,500 cells per well. After one day of growth under the conditions described above, the cells were washed twice with 100 ⁇ l of phosphate buffered saline and placed in growth arrest medium consisting of DMEM supplemented with 0.5% heat-inactivated fetal bovine serum.
  • cell number was determined by a fluorescence-based cell proliferation assay using calcein-.AM (Molecular Probes; Eugene, OR) .
  • the medium was removed and triplicate wells were incubated with ImM calcein-AM, dissolved in phosphate buffered saline, for 1 hour at 37°C. Fluorescence was determined using a Cytofluor plate reader (Millipore; Boston, MA), at 580 nm following excitement at 450 nm. Under the cell culture conditions used, there was a linear relationship between cell number (determined by Coulter counting) and fluorescence.
  • Example 1 Proliferation The proliferation of smooth muscle cells according to the assay described in Example 1 was determined in the presence of antisense polynucleotides which hybridized to portions of mouse FGF receptor 1, 2, 3 and 4, as well as an antisense polynucleotide directed against plasminogen activator inhibitor-1 (PAI-1) , as a negative control.
  • PAI-1 plasminogen activator inhibitor-1
  • Smooth muscle cells were isolated from normal human aorta or diseased human carotid artery endarterectomy specimens in order to evaluate the growth-regulated expression of FGF receptor mRNA.
  • the smooth muscle cells were obtained by enzymatic dissociation with collagenase and elastase followed by culture in DMEM supplemented with 10 percent fetal bovine serum. After 5 to 7 days of culture, the cells were plated at 40,000 cells per 100 mm 2 dish.
  • RT-PCR reverse transcriptase- polymerase chain reaction
  • GAPDH Glyceraldehyde phosphate dehydrogenase
  • FGF receptor gene 1 mRNA was undetectable in growth-arrested human smooth muscle cell cultures.
  • FGF receptor gene 1 mRNA levels were elevated in normal growth-stimulated, log phase human SMCs, but were even further stimulated in diseased growth-stimulated, log phase human SMCs.
  • the growth of SMCs derived from diseased human patients was correlated with overexpression of FGF receptor gene 1 mRNA.
  • FGFR1 + - ++ - ' The pluses represent relative band intensities of the PCR amplified products as seen on agarose gels. The minuses indicate the absence of a detectable band.
  • FGF receptor gene 1 mRNA was tested using the rat carotid artery balloon angioplasty model of restenosis. Abnormal growth of SMCs often leads to restenosis in humans; these experiments were designed to determine whether FGF receptor gene l mRNA is overexpressed in response to angioplasty.
  • the arteries were removed from the anesthetized animals, trimmed of adventitia and nerve tissue, and mRNA levels were determined by RT-PCR, according to standard procedures in the art.
  • GAPDH mRNA levels were determined as an internal control.
  • PCNA mRNA levels were determined in order to assess the proliferation of neointimal and medial SMCs.
  • FGF receptor gene 1 mRNA was induced to a maximum level of expression between 6 hours and 2 days post-angioplasty. As indicated by the expression levels of PCNA mRNA, this time frame correlates with the proliferation of medial SMCs. Table 2
  • angioplasty of the rat carotid artery induced FGF receptor 1 gene mRNA overexpression in vivo. This induction was not only maintained in in vitro cell culture, but was enriched in the population of abnormally proliferating neointimal SMCs.
  • an antisense polynucleotide directed against FGF receptor gene 1 inhibited growth of rat carotid SMCs in a dose-dependent manner.
  • an antisense polynucleotide directed against the vinculin gene CGTATGAAAC CATGGCAT, SEQ ID NO:29
  • CGTATGAAAC CATGGCAT CGTATGAAAC CATGGCAT
  • Antisense Molecules Various antisense polynucleotides were constructed in order to determine which regions of the
  • FGF receptor 1 gene were most sensitive to inhibition by antisense molecules. Inhibition studies were performed as described in Example 2. Table 4 shows the positions relative to the start codon and sequence identification numbers of the antisense polynucleotides, and their growth inhibitory effect.
  • the antisense polynucleotide designated SEQ ID NO:10 showed the highest level of growth inhibition.
  • SEQ ID NO:10 extends from -3 to +18, relative to the start codon.
  • Antisense polynucleotides directed to internal FGF receptor 1 gene sequences, such as SEQ ID NO:27 also showed appreciable growth inhibition.
  • Antisense polynucleotides directed against the human FGF receptor 1 gene, which contains a C to T substitution at position +7 (relative to the mouse sequence) were also active in inhibiting the growth of rat SMCs.
  • the similar activities of SEQ ID NO:13, directed against the mouse sequence, and SEQ ID NO:19, directed against the comparable human sequence, in inhibiting the growth of rat SMCs indicates that a one base mismatch is tolerable for target gene regulation, and suggests that overall position and length of the polynucleotide may be important determinants in efficacy of inhibition.
  • Antisense-Mediated Down Regulation of Target mRNA The specificity, efficacy and mechanism of action of antisense polynucleotides can be examined by studying the down-regulation of target mRNA by such antisense molecules.
  • growth arrested SMCs were serum-stimulated in the presence or absence of 50 ⁇ M of the antisense polynucleotide designated SEQ ID NO:10. After 3 days of contact with the antisense polynucleotide, total RNA was isolated from the SMCs, and RT-PCR was performed.
  • Table 6 shows that SEQ ID NO:10 markedly inhibited the induction of its target mRNA, while having no effect on a control housekeeping gene, GAPDH.
  • An antisense polynucleotide directed against vinculin did not affect the induction of mRNA from the FGF receptor 1 gene.
  • Example 3 The results discussed in Example 3 showed the growth-dependent overexpression of the FGF receptor 1 gene in human SMCs, and particularly those derived from diseased human patients. In these experiments, the ability of antisense polynucleotides directed against the FGF receptor gene 1 to inhibit the growth of human SMCs in culture was examined.
  • a second similar catheter that lacked a tip was filled with either an antisense molecule, or with a pharmaceutically acceptable carrier, and was attached to a syringe pump. This catheter was then inserted into the left iliac artery and advanced into the left carotid artery.
  • Antisense molecules to either FGF receptor 1 or PAI-1 (1 mM in DMEM) or the carrier alone (DMEM) was delivered at a rate of 6 ⁇ l/min for 5 min, with the catheter tied to the proximal portion of the artery to prevent blood from flowing around the catheter tip and washing the delivered material (drug or carrier) out of the artery.
  • the carotid artery was then ligated distal to the heart, near the bifurcation of the internal and external branches of the artery. After 15 min of static incubation, the ligatures and the catheter were removed to restore normal blood flow.
  • Neointimal and medial areas were measured and the intimal/medial ratio calculated. The ratio for the treatment groups was then normalized to the ratio for the control groups. The control intimal/medial ratio was 0.668 +/- 0.141.
  • Antisense polynucleotides to FGF receptor l SEQ ID NO: 10
  • the nonspecific control antisense polynucleotides to PAI-1 has no effect (see Figure 3) .
  • antisense polynucleotides directed against FGF receptor 1 mRNA specifically inhibited neointimal development in vivo in the rat carotid balloon angioplasty model of restenosis.
  • MOLECULE TYPE DNA (genomic)
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  • SEQUENCE DESCRIPTION SEQ ID NO:27: GGGGATCCTC AGGGAGCT 18
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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Abstract

The present invention is directed to a polynucleotide of about 10 to about 50 nucleic acid bases in length, which polynucleotide hybridizes to the gene encoding fibroblast growth factor receptor. The present invention is also directed to a pharmaceutical composition comprising the above polynucleotide dissolved or dispersed in a physiologically tolerable diluent. The present invention is further directed to a process for inhibiting vascular smooth muscle cell proliferation (figs. 1 and 2), a process for treating vascular smooth muscle cell proliferation (figs. 3 and 4), and a process for treating a disease state involving vascular smooth muscle cell proliferation (figs. 3 and 4), all of which processes comprise contacting vascular smooth muscle cells or administering an effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor.

Description

ANTISENSE MOLECULES DIRECTED AGAINST A FIBROBLAST GROWTH FACTOR RECEPTOR GENE FAMILY
DESCRIPTION Cross Reference to Related Application
This application is a continuation-in-part of U.S. Application Serial No. 07/999,706, filed December 31, 1992.
TECHNICAL FIELD
The present invention relates to growth-factor related polynucleotides and their use in inhibiting the proliferation of smooth muscle cells, and more specifically to antisense molecules corresponding in sequence to portions of a gene for fibroblast growth factor receptor, and their use in inhibiting the proliferation of smooth muscle cells.
BACKGROUND OF THE INVENTION Antisense polynucleotides contain artificial sequences of nucleotide bases complementary to messenger RNA (mRNA or message) or the sense strand of double stranded DNA. Admixture of sense and antisense oligo- or polynucleotides under appropriate conditions leads to binding of the two molecules, or hybridization.
When these polynucleotides bind to (hybridize with) mRNA, inhibition of translation occurs. When these polynucleotides bind to double stranded DNA, inhibition of transcription occurs. The resulting inhibition of translation and/or transcription leads to an inhibition of the synthesis of the encoded protein such as the proteins of the tissues, and more importantly here, various cellular growth factors, growth factor receptors, and oncogenes (many of which act as growth factors, receptors or mediators of signal transduction) . There are several polypeptides considered to fall into the fibroblast growth factor (FGF) family. These polypeptides, ranging in size from about 155 to about 268 amino acid residues, are angiogenic and neutrophonic molecules which are mitogenic for many cell types deriving from epithelial, esenchymal and neural origin.
Fibroblast growth factors mediate their cellular responses through binding and activation of high affinity cell surface receptors known as fibroblast growth factor receptors. To date, five such receptors have been identified and cloned from humans, and all appear to contain tyrosine kinase activity. Jaye et al., Bioch. Biophyε. Acta 1135:185-199 (1992). Activated smooth muscle cells (SMC) elaborate growth factors such as platelet derived growth factor (PDGF) , basic and acidic fibroblast growth factor, interleukins and transforming growth factor β . Likewise, the SMC increase the production of PDGF receptor, FGF receptor, and epidermal growth factor receptor.
Activation of SMC, leading to the proliferation of those cells, occurs in response to a number of stimuli, including surgical procedures such as coronary angioplasty. The proliferation of SMC results in such disease states as atherosclerosis and restenosis.
An in vitro assay system has been developed to study smooth muscle cell proliferation. This assay system is considered to be a useful model for SMC proliferation in vivo. Gordon et al. have shown that SMC proliferation results from aortic and carotid balloon catheter injury, and is a result of atherosclerosis, providing a positive correlation between SMC proliferation and stenosis. Gordon et al., Proc. Natl. Acad. Sci. USA 87:4600-4604 (1990) .
Speir et al. have studied the inhibition of SMC proliferation in vitro by using an antisense oligonucleotide to proliferating cell nuclear antigen (PCNA) . However, these workers could not inhibit proliferation below 50 %, and the inhibition required high levels of the 18-mer antisense oligonucleotide used in those studies. This invention demonstrates the biological action of antisense polynucleotides directed against the gene for fibroblast growth factor receptor, as useful for anti-proliferative activity against smooth muscle cell proliferation. This invention is applicable to a number of disease states in which the proliferation of smooth muscle cells is involved, including, but not limited to, vascular stenosis, post-angioplasty restenosis (including coronary, carotid and peripheral stenosis) , other non-angioplasty reopening procedures such as atherectomy and laser procedures, atherosclerosis, atrial-venous shunt failure, cardiac hypertrophy, vascular surgery, and organ transplant.
BRIEF SUMMARY OF THE INVENTION The present invention is directed to a polynucleotide of about 10 to about 50, preferably about 15 to about 25, and more preferably about 20, nucleic acid bases in length, which polynucleotide hybridizes to the gene encoding fibroblast growth factor receptor. A preferred polynucleotide is an antisense molecule having the sequence shown in SEQ ID NO:l.
GCACTTCCAG CCCCACAT (SEQ ID NO:l) Further preferred polynucleotides include
GCGCCCCCAG CTGACCAT (SEQ ID NO:2), GCAGGCCGGG ACTACCAT (SEQ ID NO:3), GGCCAAGAGC AGCCACAT (SEQ ID NO:4), GCACTTCCAG CTCCACAT (SEQ ID NO:5), ACGACCCCAG CTGACCAT (SEQ ID NO:6), GCAGGCAGGG GCGCCCAT (SEQ ID NO:7), GGCCAGCAGC AGCCGCAT (SEQ ID NO:8),
GCAGGCCGGG ACTACCAT (SEQ ID NO:9), GGGGATCCTC AGGGAGCT (SEQ ID NO:27), and CAGTTTCTTC TCCATTTT (SEQ ID NO:28), as well as SEQ ID NOs 10 through 21 and 26, described hereinafter.
In a preferred embodiment, the bases of the polynucleotide molecule are linked by pseudophosphate bonds that are resistant to cleavage by exonuclease and/or endonuclease enzymes. Preferred pseudophosphate bonds are phosphorothioate bonds.
The present invention is further directed to a polynucleotide of about 10 to about 50, preferably about 20 to about 40, and more preferably about 20, nucleic acid bases, which polynucleotide hybridizes to the about 5 to about 25, preferably about 10 to about 20, and more preferably about 10, nucleic acid bases flanking the start codon of the mRNA for fibroblast growth factor receptor.
A preferred such polynucleotide is an antisense molecule having the sequence shown in SEQ ID NOs 1 through 9 above, as well as SEQ ID NO:10:
GCACTTCCAG CCCCACATCC C (SEQ ID NO:10). Further preferred polynucleotides include: CTTCCAGCCC CACATCCC (SEQ ID NO:11), CCAGCCCCAC ATCCC (SEQ ID NO:12),
CTTCCAGCCC CACATCCCAG T (SEQ ID NO:13), GCACTTCCAG CCCCA (SEQ ID NO:14), CTTCCAGCCC CACAT (SEQ ID NO:15), GCCCCACATC CCAGTTCT (SEQ ID NO:16), CCAGCCCCAC ATCCCAGT (SEQ ID NO:17), GCCCCACATC CCAGT (SEQ ID NO:18), CTTCCAGCTC CACATCCCAG T (SEQ ID NO:19), CCAGCTCCAC ATCCCAGT (SEQ ID NO:20), GCTCCACATC CCAGT (SEQ ID NO:21), GAGGAGGCAC TTCCAGCTCC ACATCCC (SEQ ID NO:22),
GAGGCACTTC CAGCTCCACA TCCC (SEQ ID NO:23), GCACTTCCAG CTCCACATCC CAGT (SEQ ID NO:24), GCACTTCCAG CTCCACATCC C (SEQ ID NO:25), and CTTCCAGCTC CACATCCC (SEQ ID NO:26). The present invention is also directed to a pharmaceutical composition comprising a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor, dissolved or dispersed in a physiologically tolerable diluent.
The present invention is further directed to a process for inhibiting vascular smooth muscle cell proliferation that comprises contacting vascular smooth muscle cells whose growth is to be inhibited in an aqueous medium suitable for growth of those cells with an inhibition-effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor and maintaining said contact in said aqueous medium under biological culture conditions for a time period sufficient for the growth of the contacted cells to be inhibited.
The present invention is still further directed to a process for treating vascular smooth muscle cell proliferation that comprises administering to a host mammal in need of such treatment an effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor. The present invention is yet further directed to a process for treating a disease state involving the proliferation of vascular smooth muscle cells that comprises administering to a host mammal in need of such treatment an effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows the percentage of growth inhibition of smooth muscle cells upon the addition of various concentrations of either antisense polynucleotides directed against the gene for mouse fibroblast growth factor receptor 1, or antisense polynucleotides directed against the gene for plasminogen activator inhibitor 1.
Figure 2 shows the percentage of growth inhibition of smooth muscle cells upon the addition of either 10 μM or 50μM of antisense polynucleotides directed against the gene for mouse fibroblast growth factor 2, 3 or 4, and the gene for plasminogen activator inhibitor 1.
Figure 3 shows the percentage of growth inhibition of rat carotid artery smooth muscle cells proliferation upon the addition of either 10 μM or 50μM of antisense polynucleotides directed against mouse fibroblast growth factor gene 1, and the vinculin gene. Figure 4 shows the percentage of maximal inti al thickening of rat carotid arteries after angioplasty and treatment in vivo, upon the addition of antisense polynucleotides directed against FGF receptor 1 or PAI-1, or control treatment with carrier alone. DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to a polynucleotide of about 10 to about 50 nucleic acid bases in length, which polynucleotide hybridizes to the gene for fibroblast growth factor receptor.
The gene for fibroblast growth factor receptor can be derived from any mammal, including mouse and humans. Preferably, the gene for fibroblast growth factor receptor is that of human fibroblast growth factor receptor.
The polynucleotide may preferably be from about 15 to about 25 nucleic acid bases in length, and more preferably about 20 nucleic acid bases in length.
The present invention is further directed to a polynucleotide of about 10 to about 50, preferably about 20 to about 40, and more preferably about 20, nucleic acid bases, which polynucleotide hybridizes to the about 5 to about 25, preferably about 10 to about 20, and more preferably about 10, nucleic acid bases flanking the start codon of the mRNA for fibroblast growth factor receptor.
It is to be understood that the present invention contemplates a polynucleotide that hybridizes to any of the genes encoding a member of the fibroblast growth factor receptor family. Any such polynucleotide capable of inhibiting the proliferation of smooth muscle cell proliferation can be used.
As used herein, "polynucleotide" refers to a covalently linked sequence of nucleotides in which the 3# position of the pentose of one nucleotide is joined by a phosphodiester group to the 5' position of the pentose of the next nucleotide. The nucleotides may be composed of deoxyribonucleotides or ribonucleotides.
Polynucleotide hybridization of greater than about 90 percent homology (identity) , and more preferably about 99 percent homology, is contemplated in the present invention.
A. The Polynucleotides A preferred polynucleotide is an antisense molecule having the sequence shown in SEQ ID N0:1, directed against the gene for mouse fibroblast growth factor (FGF) receptor 1.
GCACTTCCAG CCCCACAT (SEQ ID N0:1J Further preferred polynucleotides include
GCGCCCCCAG CTGACCAT (SEQ ID NO: 2 ] , directed against the gene for mouse FGF receptor 2,
GCAGGCCGGG ACTACCAT (SEQ ID NO:3] , directed against the gene for mouse FGF receptor 3, GGCCAAGAGC AGCCACAT (SEQ ID NO: ] , directed against the gene for mouse FGF receptor 4,
GCACTTCCAG CTCCACAT (SEQ ID NO:5] , directed against the gene for human FGF receptor 1, ACGACCCCAG CTGACCAT (SEQ ID NO:6] , directed against the gene for human FGF receptor 2,
GCAGGCAGGG GCGCCCAT (SEQ ID NO: 1 ) , directed against the gene for human FGF receptor 3,
GGCCAGCAGC AGCCGCAT (SEQ ID NO:8] , directed against the gene for human FGF receptor 4, and GCAGGCCGGG ACTACCAT (SEQ ID NO:9] , directed against the gene for human FGF receptor 5.
The sequence of the polynucleotide directed against the gene for human FGF receptor 5 shown in SEQ ID NO:9 is identical to the polynucleotide directed against the gene for mouse FGF receptor 3 shown in SEQ ID NO:3; however, different SEQ ID NOs will be used to avoid confusion and because of the fact that the human and mouse sequences differ in other locations.
Further preferred polynucleotides include GGGGATCCTC AGGGAGCT (SEQ ID NO:27) , and CAGTTTCTTC TCCATTTT (SEQ ID NO:28), both of which are directed against internal sequences of the mouse FGF receptor 1 gene,
GCACTTCCAG CCCCACATCC C (SEQ ID NO:10), directed against position -3 to +18, relative to the start codon, of mouse FGF receptor gene 1,
CTTCCAGCCC CACATCCC (SEQ ID NO:11), directed against position -3 to +15, relative to the start codon, of mouse FGF receptor gene 1, CCAGCCCCAC ATCCC (SEQ ID NO:12), directed against position -3 to +12, relative to the start codon, of mouse FGF receptor gene 1,
CTTCCAGCCC CACATCCCAG T (SEQ ID NO:13), directed against position -6 to +15, relative to the start codon, of mouse FGF receptor gene 1,
GCACTTCCAG CCCCA (SEQ ID NO:14), directed against position +4 to +18, relative to the start codon, of mouse FGF receptor gene 1,
CTTCCAGCCC CACAT (SEQ ID NO:15), directed against position +1 to +15, relative to the start codon, of mouse FGF receptor gene 1,
GCCCCACATC CCAGTTCT (SEQ ID NO:16), directed against position -9 to +9, relative to the start codon, of mouse FGF receptor gene 1, CCAGCCCCAC ATCCCAGT (SEQ ID NO:17), directed against position -6 to +12, relative to the start codon, of mouse FGF receptor gene 1,
GCCCCACATC CCAGT (SEQ ID NO:18), directed against position -6 to +9, relative to the start codon, of mouse FGF receptor gene 1,
CTTCCAGCTC CACATCCCAG T (SEQ ID NO:19), directed against position -6 to +15, relative to the start codon, of human FGF receptor gene 1, CCAGCTCCAC ATCCCAGT (SEQ ID NO:20), directed against position -6 to +12, relative to the start codon, of human FGF receptor gene 1,
GCTCCACATC CCAGT (SEQ ID NO:21) , directed against position -6 to +9, relative to the start codon, of human FGF receptor gene 1
GAGGAGGCAC TTCCAGCTCC ACATCCC (SEQ ID NO:22), directed against position -3 to +24, relative to the start codon of human FGF receptor gene 1, GAGGCACTTC CAGCTCCACA TCCC (SEQ ID NO:23), directed against position -3 to +21, relative to the start codon of human FGF receptor gene 1,
GCACTTCCAG CTCCACATCC CAGT (SEQ ID NO:24), directed against position -6 to +18, relative to the start codon of human FGF receptor gene 1,
GCACTTCCAG CTCCACATCC C (SEQ ID NO:25) , directed against position -3 to +18, relative to the start codon of human FGF receptor gene 1, and
CTTCCAGCTC CACATCCC (SEQ ID NO:26) , directed against position -6 to +9, relative to the start codon of human FGF receptor gene 1.
In a preferred embodiment, the bases of the polynucleotide, e.g., SEQ ID N0:1, are linked by pseudophosphate bonds that are resistant to cleavage by exonuclease or endonuclease enzymes. Exonuclease enzymes hydrolyze the terminal phosphodiester bond of a nucleic acid. Endonuclease enzymes hydrolyze internal phosphodiester bonds of a nucleic acid.
By replacing a phosphodiester bond with one that is resistant to the action of exonucleases or endonucleases, the stability of the nucleic acid in the presence of those exonucleases or endonucleases is increased. As used herein, pseudophosphate bonds include, but are not limited to, methylphosphonate, phosphomorpholidate, phosphorothioate, phosphorodithioate and phosphoroselenoate bonds. Additionally, exonuclease and/or endonuclease resistant polynucleotides can be obtained by blocking the 3 ' and/or 5' terminal nucleotides with substituent groups such as acridine, cholesterol or a methyl group.
Preferred pseudophosphate bonds are phosphorothioate bonds. The pseudophosphate bonds may comprise the bonds at the 3' and or 5' terminus, the bonds from about 1 to about 5 of the 3' and/or 5' terminus bases, or the bonds of the entire polynucleotide. A preferred polynucleotide with pseudophosphate bonds is one in which all of the bonds are comprised of pseudophosphate bonds.
DNA or RNA polynucleotides can be prepared using several different methods, as is well known in the art. See, e.g., Ausubel et al. (eds.). Current Protocols in Molecular Biology, John Wiley & Sons, New York (1990) . The phosphoramidate synthesis method is described in Caruthers et al., Meth. Enzy ol. 154:287 (1987) ; the phosphorothioate polynucleotide synthesis method is described in Iyer et al., J. Am. Chem. Soc. 112:1253 (1990).
The present invention is also directed to a pharmaceutical composition comprising a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor, dissolved or dispersed in a physiologically tolerable diluent. Preferably, the polynucleotide is from about 15 to about 25 nucleic acid bases in length, and more preferably about 20 nucleic acid bases in length.
The present invention includes one or more polynucleotides as described above formulated into compositions together with one or more non-toxic physiologically tolerable or acceptable diluents. carriers, adjuvants or vehicles that are collectively referred to herein as diluents, for parenteral injection, for oral administration in solid or liquid form, for rectal or topical administration, or the like. The compositions can be administered to humans and animals either orally, rectally, parenterally (intravenous, by intramuscularly or subcutaneously) , intracisternally, intravaginally, intraperitoneally, locally (powders, ointments or drops) , or as a buccal or nasal spray.
The compositions can also be delivered through a catheter for local delivery at the site of vascular damage, via an intracoronary catheter or stent (a tubular device composed of a fine wire mesh) , or via a biodegradable polymer. The compositions may also be complexed to ligands, such as antibodies, for targeted delivery of the compositions to the site of smooth muscle cell proliferation.
The compositions are preferably administered via parenteral delivery at the local site of smooth muscle cell proliferation. The parenteral delivery is preferably via catheter.
Compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like) , suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
These compositions can also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
Besides such inert diluents, the composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
Suspensions, in addition to the active compounds, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like. Dosage forms for topical administration of a compound of this invention include ointments, powders, sprays and inhalants. The active component is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers or propellants as may be required. Ophthalmic formulations, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.
Actual dosage levels of active ingredients in the compositions of the present invention may be varied so as to obtain an amount of active ingredient that is effective to obtain a desired therapeutic response for a particular composition and method of administration. The selected dosage level therefore depends upon the desired therapeutic effect, on the route of administration, on the desired duration of treatment and other factors.
The total daily dose of the compounds of this invention administered to a host in single or divided doses may be in amounts, for example, of from about 1 nanomol to about 5 micromols per kilogram of body weight. Dosage unit compositions may contain such amounts of such submultiples thereof as may be used to make up the daily dose. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the body weight, general health, sex, diet, time and route of administration, rates of absorption and excretion, combination with other drugs and the severity of the particular disease being treated.
B. The Processes
The present invention is further directed to a process for inhibiting vascular smooth muscle cell proliferation that comprises contacting vascular smooth muscle cells whose growth is to be inhibited in an aqueous medium suitable for growth of those cells with an inhibition-effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor and maintaining said contact in said aqueous medium under biological culture conditions for a time period sufficient for the growth of the contacted cells to be inhibited. As used herein, an "inhibition-effective amount" is that amount of a polynucleotide of the present invention which is sufficient for inhibiting the growth or killing a cell contacted with such a polynucleotide. Means for determining an inhibition- effective amount in a particular subject will depend, as is well known in the art, on the nature of the polynucleotide used, the mass of the subject being treated, whether killing or growth inhibition of the cells is desired, and the like.
Contact is achieved by admixing the composition with a preparation of vascular smooth muscle cells.
Biological culture conditions are those conditions necessary to maintain the growth and replication of the vascular smooth muscle cells in a normal, polynucleotide-free environment. These biological culture conditions, encompassing such factors as temperature, humidity, atmosphere, pH and the like, must be suitable for the proliferation of vascular smooth muscle cells in the absence of polynucleotides so that the effects of such polynucleotides on relevant growth parameters can be measured.
A preferred polynucleotide useful in this process has the sequence shown in SEQ ID N0:1. A further preferred polynucleotide useful in this process links the bases of SEQ ID NO:l by pseudophosphate bonds that are resistant to cleavage by exonuclease enzymes. Preferred pseudophosphate bonds are phosphorothioate bonds.
Further preferred polynucleotides have the sequences shown in SEQ ID NO:2 through 28.
The present invention is still further directed to a process for treating vascular smooth muscle cell proliferation that comprises administering to a host mammal in need of such treatment an effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor.
A host mammal in need of the treatment of a process for the inhibition of vascular smooth muscle cell proliferation suffers from a disease state in which such proliferation is implicated. Such disease states include vascular stenosis, post-angioplasty restenosis (including coronary, carotid and peripheral stenosis) , other non-angioplasty reopening procedures such as atherectomy and laser procedures, atherosclerosis, atrial-venous shunt failure, cardiac hypertrophy, vascular surgery, by-pass surgery and organ transplant.
In a preferred embodiment, the polynucleotide as described above is dissolved or dispersed in a physiologically tolerable diluent.
The present invention is yet further directed to a process for treating a disease state involving the proliferation of vascular smooth muscle cells that comprises administering to a host mammal in need of such treatment an effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor.
A disease state involving the proliferation of vascular smooth muscle cells include, but not limited to, vascular stenosis, post-angioplasty restenosis (including coronary, carotid and peripheral stenosis) , other non-angioplasty reopening procedures such as atherectomy and laser procedures, atherosclerosis, atrial-venous shunt failure, cardiac hypertrophy, vascular surgery, and organ transplant. The following examples further illustrate the invention and are not to be construed as limiting of the specification and claims in any way.
EXAMPLES
Materials and Methods
Antisense Polynucleotide Preparation All reagents were from Eppendorf or Glen Research. Antisense polydeoxynucleotide solid phase syntheses were performed on Millipore CPG columns using cyanoethyl phosphoramidite chemistry on an Eppendorf Synostat D300 DNA synthesizer replacing iodine by 3H- l,2-benzodithiol-3-one 1,1-dioxide (BDTD Beaucage reagent) . The polynucleotide was cleaved from the solid support by incubation with 3 ml of fresh, concentrated (30%) ammonium hydroxide for 90 minutes. Cleavage was facilitated by mixing of the solution every 30 minutes with the help of two 5 ml slip-tip syringes. The solution was collected in a screw-capped glass vial and deprotection was accomplished either at room temperature for 24 hours or at 55 C for 5 hours. The contents were transferred to a 13x100 mm glass tube, chilled on ice and evaporated to dryness using a Savant Speed-Vac.
The polynucleotide was then dissolved in 1 ml of 0.1M triethylammonium acetate (TEAA) , pH 7.0. The oligo was detritylated and purified on a Rainin Dyna ax C18 semipreparative column (10mm x 25cm, 5um, 300 A) . The mobile phases were (A): 0.1M TEAA, pH7.0, 5% acetonitrile; (B) : 95% acetonitrile, 5% water; (C) : 0.5% TFA in water. The column was developed at 2ml/min with the following gradient: 10% B in A, 10 min; 100% A, 4 min; 100% C, 8 min; 100% A, 8 min; 100% A to 45% B in 24 min. This procedure first separates the trityl-on full length polynucleotide from its failure sequences containing free hydroxyl groups and synthesis reagents. This is followed by the removal of 5'-DMT by 0.5% TFA. Finally the gradient resolved the desired detritylated sequence from other contaminants.
Absorbance was monitored at 260mm to identify fractions containing the polynucleotide which was then evaporated. The polynucleotide was dissolved in 1 ml water, evaporated to remove volatile salts, and finally dissolved in 0.5 ml sterile, low TE (lOmM Tris, ImM EDTA, pH 7.5) .
The polynucleotide concentration was determined by measuring the absorbance at 260 nm. Typical yields were 30-40%. The integrity of the polynucleotide was determined by polyacrylamide gel electrophoresis (PAGE; 20% polyacrylamide, 7M urea) and staining with 0.2% methylene blue.
Smooth Muscle Cell Isolation and Culture Male Sprague-Dawley rats weighing 350-450 g were euthanized with carbon dioxide. The carotid arteries were removed and trimmed free of adventitia, nerve, and fat under a dissecting microscope. Arteries were cut into approximately 1mm3 pieces and placed in a 125 ml Erlenmeyer flask containing 0.67 ml/carotid artery of the following enzyme cocktail:
79.2 ml Hanks Balanced Salt Solution (Gibco; HBSS), 0.8 ml 0.2M CaCl2 (Fisher), 0.286 g HEPES free acid (Calbiochem), 0.03 g trypsin inhibitor (Sigma; Type I-S; 10,000 units/mg) , 0.16 g bovine serum albumin
(Sigma; Fraction V) , 600 units elastase (Sigma; Type II- A; 28 units/mg), 16,000 units collagenase (Worthington; CLS II; 353 units/mg) adjusted to pH7.4, and 0.2μm filtered. The flask was placed on an orbital shaker at 150 rpm at 37°C for 2-2.5 hr. The suspension was triturated vigorously and filtered through a 70 μm nylon cell strainer. The filtrate was then centrifuged at 400 x g for 10 min. The pellet was resuspended in 4 ml/carotid of the following media: 20% fetal bovine serum albumin (Hyclone; FBS) ; 2mM glutamine (Gibco) ; 100 units/ml penicillin G sodium (Gibco) ; 100 ug/ml streptomycin sulfate (Gibco) ; DMEM (Gibco) . The cell suspension from one carotid was then seeded into one T25 flask (Falcon) and maintained at 37βC in 5% C02.
Proliferation Assay After 6-7 days, cells were rinsed twice with
PBS (phosphate buffered saline) and harvested by the addition of 4 ml of 0.05% trypsin-EDTA (Gibco; 0.25% trypsin-EDTA) followed by incubation at 37*C for 3-5 min. The flask was rinsed with an additional 4 ml media (DMEM, 20% PBS, 2 mM glutamine, 50 units/ml penicillin, 50 ug/ml streptomycin) . The trypsinized cells and the rinse were combined and centrifuged at 400 x g for 10 min.
The supernatant was removed and 5 is of fresh media was added to the pellet. The pellet was resuspended by vigorous trituration, and the number of cells was determined using a Coulter counter.
The cells were diluted to 3,500 cells/100 ul and, using a 12 channel digital micropipette, '100 ul/well of the cells were seeded in a 96 well (Falcon) flat-bottom, microtiter cell culture plate. The culture plate was then incubated at 37*C in 5% C02.
The following day, each well was rinsed twice with 100 ul PBS, and overlaid with 100 ul/well growth arrest media: 0.1% FBS (heat inactivated at 65#C for 45 min.); 2mM glutamine; 50 units/ml penicillin; 50 ug/ml streptomycin.
Four days later, the growth arrest media was removed. The cell number was determined (treatment day counts) using a Coulter counter by averaging the cell number from three wells. To the remaining wells was added 100 ul complete media (DMEM, 10% FBS/65'C inactivated, glutamine, pen/strep) without or with antisense polynucleotides and the plates were placed in an incubator at 37*C in 5% C02.
Three days later, the wells were rinsed twice with 100 ul each PBS. The cell number from 3 wells was again determined (assay day counts) using a Coulter counter. To the remaining wells was added 100 μl of 45 μg/ml calcein A-M (Molecular Probes) in PBS. The plates were incubated for 1 hr. at 37*C.
After incubation, fluorescence was determined using a Cytofluor 2350 (Millipore) microtiter plate reader with excitation at 485 nm and emission at 530 nm. Growth in cell number was calculated by subtracting treatment day cell counts from assay day cell counts. Based on the established linear relationship between fluorescence and cell number, the percent growth inhibition by the antisense polynucleotides was determined.
In Vivo Efficacy Experiments Male Sprague-Dawley rats weighing 375-425 g were housed for at least 5 days prior to surgery. Anesthesia was performed with 2-2.5% inhalation of isofluorene; all surgery was performed under sterile conditions.
Cutdowns were performed on the carotid and iliac arteries and 25% bupiviamine used as a topical anesthetic. A 2F embolectomy catheter was inserted into the left iliac artery and advanced to the distal end of the left carotid artery. The balloon was inflated and pulled down the artery 3 times. The catheter was then removed. A second similar catheter that lacked a tip, was filled with antisense polynucleotide (drug) or diluent (vehicle) , and was attached to a syringe pump. This catheter was then inserted into the left iliac and advanced into the left carotid. .Antisense polynucleotide or vehicle was then delivered at 6 μl/min for 5 min with the catheter tied to the proximal portion of the artery to prevent blood from flowing around the catheter tip and washing the drug out of the artery. The carotid was then ligated distal to the heart near the bifurcation of the internal and external branches of the artery. After 15 min of static incubation, the ligatures and the catheter were removed to restore normal blood flow.
Fifty ul of oligo or vehicle was then applied to the adventitial surface of the carotid and 150 units/kg of i.v. heparin was delivered.
The two incisions were then closed with 4.0 silk suture. Rats recovered under an infrared heating lamp connected to a proportional regulated heater. Topical antibiotics were applied to the incisions.
Two weeks later, rats were sacrificed with C02 and the abdominal aorta perfused retrogradely at 3 ml/min with heparinized PBS (10 units/ml) to remove the blood. Perfusion was then performed with 10% formalin at 2.5 ml/min for 15 min. These flow rates result in carotid artery pressures of about 100 mm Hg.
The treated section of the left carotid artery was removed and the central portion embedded in paraffin, cut in 5 μm sections, and stained with hematoxylin and eosin. A frame grabber (Targa+) and image analysis software (Java) were used to measure cross-sectional areas of the medial and intimal layers. To standardize the intimal area for arteries of different size, the intimal area was divided by the medial area and this ratio used as the parameter to compare drug treatment with control.
Example 1. SMC Proliferation Assay
Carotid arteries were dissected from male Sprague-Dawley rats weighing 200 to 300 grams.
Approximately 1 mm3 minces were incubated in Dulbecco's minimal essential medium (DMEM) supplemented with 20 percent fetal bovine serum. The medium was changed every three to four days. After 2 to 2.5 weeks of growth, trypsin was added to the growth culture to isolate cells which had grown out of the arterial explant. These isolated cells were plated in 96 well trays at a concentration of 2,500 cells per well. After one day of growth under the conditions described above, the cells were washed twice with 100 μl of phosphate buffered saline and placed in growth arrest medium consisting of DMEM supplemented with 0.5% heat-inactivated fetal bovine serum.
After four days in growth arrest medium, cell number was determined by a fluorescence-based cell proliferation assay using calcein-.AM (Molecular Probes; Eugene, OR) . The medium was removed and triplicate wells were incubated with ImM calcein-AM, dissolved in phosphate buffered saline, for 1 hour at 37°C. Fluorescence was determined using a Cytofluor plate reader (Millipore; Boston, MA), at 580 nm following excitement at 450 nm. Under the cell culture conditions used, there was a linear relationship between cell number (determined by Coulter counting) and fluorescence. Additional sets of triplicate wells were either untreated (controls) or treated with the indicated concentrations of polynucleotides dissolved in DMEM with 10% serum heat-inactivated at 65°C. After an additional three days, fluorescence was again determined using calcein-AM, as described above.
Example 2. Polynucleotide Effects on SMC
Proliferation The proliferation of smooth muscle cells according to the assay described in Example 1 was determined in the presence of antisense polynucleotides which hybridized to portions of mouse FGF receptor 1, 2, 3 and 4, as well as an antisense polynucleotide directed against plasminogen activator inhibitor-1 (PAI-1) , as a negative control.
The results of these assays are shown in Figures 1 and 2. The data in the bar graphs is depicted to indicate that 100% inhibition reflects the absence of SMC proliferation during the assay. Values of less than 0% indicate that the treated cells proliferated to a greater extent than did untreated cells. Values of greater than 100% means that there were fewer cells at the end of the assay than at the beginning.
The data show that 50 μM of the antisense polynucleotide against mouse FGF receptor 1 showed nearly 75% inhibition of SMC proliferation, compared to about 10% inhibition by the control antisense polynucleotide directed against PAI-1. See Figure 1. The data in Figure 2 show that 50 μM of the antisense polynucleotides directed against FGF receptors 2, 3 and 4 showed between 40 and 80 percent inhibition of SMC proliferation, compared to about 10% inhibition by the control polynucleotide. Example 3. Growth-regulated Expression of FGF
Receptor mRNA in Humans Smooth muscle cells were isolated from normal human aorta or diseased human carotid artery endarterectomy specimens in order to evaluate the growth-regulated expression of FGF receptor mRNA. The smooth muscle cells were obtained by enzymatic dissociation with collagenase and elastase followed by culture in DMEM supplemented with 10 percent fetal bovine serum. After 5 to 7 days of culture, the cells were plated at 40,000 cells per 100 mm2 dish. The day after plating, the cells were growth-stimulated by the removal of the growth medium and addition of DMEM plus 10 percent fetal bovine serum, or growth-arrested by the removal of the growth medium and the addition of DMEM plus 0.1 percent fetal bovine serum. Three days after growth-stimulation or growth-arrest, total cellular RNA was isolated according to standard protocols, and mRNA levels were determined using reverse transcriptase- polymerase chain reaction (RT-PCR) , again according to standard protocols.
Glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA levels were also determined as an internal control for these experiments. GAPDH is a housekeeping gene whose expression would be expected to remain constant in the tested cells. As the data in Table 1 show, GAPDH levels remained constant.
As further shown in the data in Table 1, below, FGF receptor gene 1 mRNA was undetectable in growth-arrested human smooth muscle cell cultures. FGF receptor gene 1 mRNA levels were elevated in normal growth-stimulated, log phase human SMCs, but were even further stimulated in diseased growth-stimulated, log phase human SMCs. Hence, the growth of SMCs derived from diseased human patients was correlated with overexpression of FGF receptor gene 1 mRNA.
Table 1 Normal Aorta Carotid Endarterectomv* mRNA Log Arrest Log Arrest GAPDH +++++ +++++ +++++ +++++
FGFR1 + - ++ - 'The pluses represent relative band intensities of the PCR amplified products as seen on agarose gels. The minuses indicate the absence of a detectable band.
Example . Overexpression of FGF Receptor mRNA After
Angioplastv The overexpression of FGF receptor gene 1 mRNA was tested using the rat carotid artery balloon angioplasty model of restenosis. Abnormal growth of SMCs often leads to restenosis in humans; these experiments were designed to determine whether FGF receptor gene l mRNA is overexpressed in response to angioplasty.
At various times after balloon angioplasty of the rats, the arteries were removed from the anesthetized animals, trimmed of adventitia and nerve tissue, and mRNA levels were determined by RT-PCR, according to standard procedures in the art.
As in Example 3, GAPDH mRNA levels were determined as an internal control. PCNA mRNA levels were determined in order to assess the proliferation of neointimal and medial SMCs.
As shown in Table 2, below, FGF receptor gene 1 mRNA was induced to a maximum level of expression between 6 hours and 2 days post-angioplasty. As indicated by the expression levels of PCNA mRNA, this time frame correlates with the proliferation of medial SMCs. Table 2
I
Time Post-Angioplasty* f mRNA Control 2 hr 4 hr 6 hr 1 d 2 d 4 d 7 d 14 d
GAPDH ++ ++ ++ +++ +++ +++ +++ +++ +++
FGFR1 + - + ++++ ++++ ++++ ++ - +++
PCNA - - - + +++ ++++ nd" - ++
'Values represent relative band intensities of amplified products on gels, 10 "Not determined.
SMCs were then isolated from rat arteries after angioplasty according to the technique described in Example 3. Expression levels of FGF receptor gene 1, PCNA and GAPDH mRNAs were again determined by RT-PCR, and the results are shown in Table 3. The altered in vivo genotype observed in the arteries was maintained in tissue culture. FGF receptor gene 1 mRNA was overexpressed in both neointimal and medial SMCs in vitro, with greater overexpression in the neointimal SMCs. As the expression levels of PCNA mRNA show, neointimal cells were also more actively proliferating than were medial cells.
Table 3 mRNA Neointimal SMCs Medial SMCs
GAPDH +++++ +++++
FGFR1 +++ ++
PCNA ++++ +++
Thus, angioplasty of the rat carotid artery induced FGF receptor 1 gene mRNA overexpression in vivo. This induction was not only maintained in in vitro cell culture, but was enriched in the population of abnormally proliferating neointimal SMCs.
Example 5. Effects of FGF Receptor Antisense
Molecule on Rat Carotid Artery SMCs To determine if a relationship exists between
SMC growth and FGF receptor gene 1 mRNA expression, the effects of an antisense oligonucleotide to the FGF receptor gene 1 on the growth of SMCs isolated from rat carotid arteries was examined, as described in Example 2.
As shown in Figure 3, an antisense polynucleotide directed against FGF receptor gene 1 (SEQ ID NO: 1) inhibited growth of rat carotid SMCs in a dose-dependent manner. In contrast, an antisense polynucleotide directed against the vinculin gene (CGTATGAAAC CATGGCAT, SEQ ID NO:29) exhibited relatively little growth inhibition.
Example 6. Seguence Optimization of FGF Receptor
Antisense Molecules Various antisense polynucleotides were constructed in order to determine which regions of the
FGF receptor 1 gene were most sensitive to inhibition by antisense molecules. Inhibition studies were performed as described in Example 2. Table 4 shows the positions relative to the start codon and sequence identification numbers of the antisense polynucleotides, and their growth inhibitory effect.
Position SEO ID NO % Growth Inhibition
Mouse FGFR1 Sequences
-3/18 10 90
-3/15 11 77
-3/12 12 57
+1/18 1 52
-6/15 13 50
+4/18 14 46
+1/15 15 46
-9/9 16 40
-6/12 17 39
-6/9 18 27
Internal Mouse FGFR1 Sequences
27 71
28 48
Human FGFR1 Sequences
-6/15 19 63
-6/12 20 44
-6/9 21 11
-3/24 22 79
-3/21 23 65
-6/18 24 64
-3/18 25 58
-3/15 26 41 Vinculin
+1/18 29 15
As the data show, the antisense polynucleotide designated SEQ ID NO:10 showed the highest level of growth inhibition. SEQ ID NO:10 extends from -3 to +18, relative to the start codon. Antisense polynucleotides directed to internal FGF receptor 1 gene sequences, such as SEQ ID NO:27 also showed appreciable growth inhibition.
Antisense polynucleotides directed against the human FGF receptor 1 gene, which contains a C to T substitution at position +7 (relative to the mouse sequence) were also active in inhibiting the growth of rat SMCs. The similar activities of SEQ ID NO:13, directed against the mouse sequence, and SEQ ID NO:19, directed against the comparable human sequence, in inhibiting the growth of rat SMCs indicates that a one base mismatch is tolerable for target gene regulation, and suggests that overall position and length of the polynucleotide may be important determinants in efficacy of inhibition.
Antisense polynucleotides directed against regions surrounding the start site of other members of the mouse FGF receptor family also exhibited significant inhibition of rat SMCs. Table 5 shows the results of these studies.
Table 5 Name Position SEQ ID NO % Growth Inhibition
FGFR1 +1/18 1 52
FGFR2 +1/18 2 41
FGFR3 +1/18 3 47
FGFR4 +1/18 4 40
6. Antisense-Mediated Down Regulation of Target mRNA The specificity, efficacy and mechanism of action of antisense polynucleotides can be examined by studying the down-regulation of target mRNA by such antisense molecules. In these experiments, growth arrested SMCs were serum-stimulated in the presence or absence of 50 μM of the antisense polynucleotide designated SEQ ID NO:10. After 3 days of contact with the antisense polynucleotide, total RNA was isolated from the SMCs, and RT-PCR was performed.
Table 6 shows that SEQ ID NO:10 markedly inhibited the induction of its target mRNA, while having no effect on a control housekeeping gene, GAPDH. An antisense polynucleotide directed against vinculin did not affect the induction of mRNA from the FGF receptor 1 gene. These data suggest that the growth inhibition by antisense polynucleotides directed against the FGF receptor 1 gene are the result of specific inhibition of the production of FGF receptor 1 mRNA.
Table 6
Antisense % Growth Days of Serum Stimulation Treatment Inhibition mRNA 0 3
None 0 GAPDH ++++ ++++
None 0 FGFR1 +++ ++++ SEQ ID NO:10 85 GAPDH ++++ ++++ SEQ ID NO:10 85 FGFR1 +++ + Vinculin 18 GAPDH ++++ ++++ Vinculin 18 FGFR1 +++ ++++
Example 7. Antisense Inhibition of Human SMCs in
Culture
The results discussed in Example 3 showed the growth-dependent overexpression of the FGF receptor 1 gene in human SMCs, and particularly those derived from diseased human patients. In these experiments, the ability of antisense polynucleotides directed against the FGF receptor gene 1 to inhibit the growth of human SMCs in culture was examined.
Human SMCs were grown essentially as described in Example 3. Table 7 shows that SEQ ID NO:10, which is 94% homologous with the human FGF receptor gene 1 sequence, inhibited proliferation of SMCs from both normal human aorta and diseased human endarterectomies. RT-PCR analysis of total RNA shows that this inhibition is caused by specific down regulation of FGF receptor gene 1 mRNA by the antisense polynucleotide SEQ ID NO:10. These data are shown in Table 8.
Table 7
Cell Source SEQ ID NO:10* Vinculin* Aorta 90 9
Endarterectomy 100 12
'These values represent % growth inhibition at 50 μM antisense.
Table 8 Antisense % Growth mRNA
Treatment Inhibition GAPDH FGFR1 None 0 +++++ +++
SEQ ID NO:10 84 +++++ +
Example 8. Effects of FGF Receptor Antisense
Molecules on Rat Carotid Arteries In Vivo Balloon angioplasty of the rat carotid artery was performed essentially as described by Clowes et al. (Lab. Invest. 4_ :327-33 (1983)). Briefly, male Sprague- Dawley rats weighing 375-425 g were anesthetized. A 2F embolectomy catheter was then inserted into the left iliac artery and advanced to the distal end of the left carotid artery. The balloon was inflated and pulled down the artery three times. The catheter was then removed.
A second similar catheter that lacked a tip was filled with either an antisense molecule, or with a pharmaceutically acceptable carrier, and was attached to a syringe pump. This catheter was then inserted into the left iliac artery and advanced into the left carotid artery.
Antisense molecules to either FGF receptor 1 or PAI-1 (1 mM in DMEM) or the carrier alone (DMEM) was delivered at a rate of 6 μl/min for 5 min, with the catheter tied to the proximal portion of the artery to prevent blood from flowing around the catheter tip and washing the delivered material (drug or carrier) out of the artery. The carotid artery was then ligated distal to the heart, near the bifurcation of the internal and external branches of the artery. After 15 min of static incubation, the ligatures and the catheter were removed to restore normal blood flow.
Fifty μl of antisense polynucleotides, or carrier, was then applied to the adventitial surface of the carotid artery.
Two weeks after this treatment, the animals were sacrificed, perfuse-fixed with 10% formalin, and the central portion of the carotid artery was embedded in paraffin, according to standard protocols. Five μm sections were then stained with hematoxylin and eosin, again according to standard protocols.
Neointimal and medial areas were measured and the intimal/medial ratio calculated. The ratio for the treatment groups was then normalized to the ratio for the control groups. The control intimal/medial ratio was 0.668 +/- 0.141. Antisense polynucleotides to FGF receptor l (SEQ ID NO: 10) inhibited intimal thickening by approximately 50% (intimal/medial ratio of 0.346 +/- 0.043), while the nonspecific control antisense polynucleotides to PAI-1 has no effect (see Figure 3) . Thus, antisense polynucleotides directed against FGF receptor 1 mRNA specifically inhibited neointimal development in vivo in the rat carotid balloon angioplasty model of restenosis.
The foregoing specification, including the specific embodiments and examples is intended to be illustrative of the present invention and is not to be taken as limiting. Numerous other variations and modifications can be effected without departing from the true spirit and scope of the present invention. SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Denner, Larry A. Rege, Ajay A. Dixon, Richard A.F.
(ii) TITLE OF INVENTION: ANTISENSE MOLECULES DIRECTED AGAINST A FIBROBLAST GROWTH FACTOR RECEPTOR GENE FAMILY
(iii) NUMBER OF SEQUENCES: 29
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(A) APPLICATION NUMBER: US 07/999,706
(B) FILING DATE: December 31, 1992
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GCACTTCCAG CCCCACAT 18
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(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: CTTCCAGCCC CACAT 15
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: GCCCCACATC CCAGTTCT 18
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: CCAGCCCCAC ATCCCAGT 18
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: GCCCCACATC CCAGT 15
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: CTTCCAGCTC CACATCCCAG T 21
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: CCAGCTCCAC ATCCCAGT 18
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: GCTCCACATC CCAGT 15
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: GAGGAGGCAC TTCCAGCTCC ACATCCC 27
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: GAGGCACTTC CAGCTCCACA TCCC 24 (2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: GCACTTCCAG CTCCACATCC CAGT 24
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: GCACTTCCAG CTCCACATCC C 21
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: CTTCCAGCTC CACATCCC 18
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: GGGGATCCTC AGGGAGCT 18
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: CAGTTTCTTC TCCATTTT 18
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: CGTATGAAAC CATGGCAT 18

Claims

1. A polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor.
2. The polynucleotide of claim 1 wherein said gene for fibroblast growth factor receptor is human fibroblast growth factor receptor.
3. The polynucleotide of claim 1 that is an antisense molecule having the sequence shown in SEQ ID N0:1 through 24.
4. The polynucleotide of claim 1 wherein the bases of said polynucleotide are linked by pseudophosphate bonds that are resistant to cleavage by exonuclease or endonuclease enzymes.
5. The polynucleotide of claim 4 wherein said bonds are phosphorothioate bonds.
6. A pharmaceutical composition comprising a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor, dissolved or dispersed in a physiologically tolerable diluent.
7. The pharmaceutical composition of claim 6 wherein the polynucleotide that is an antisense molecule having the sequence shown in SEQ ID N0:1 through 24.
8. The pharmaceutical composition of claim 6 wherein the bases of said polynucleotide are linked by pseudophosphate bonds that are resistant to cleavage by exonuclease or endonuclease enzymes.
9. The pharmaceutical composition of claim 8 wherein said bonds are phosphorothioate bonds.
10. A process for inhibiting vascular smooth muscle cell proliferation that comprises contacting vascular smooth muscle cells whose growth is to be inhibited in an aqueous medium suitable for growth of those cells with an inhibition-effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor and maintaining said contact in said aqueous medium under biological culture conditions for a time period sufficient for the growth of the contacted cells to be inhibited.
11. A process for treating vascular smooth muscle cell proliferation that comprises administering to a host mammal in need of such treatment an effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor.
12. The process of claim 11 wherein said polynucleotide is dissolved or dispersed in a physiologically tolerable diluent.
13. The process of claim 11 wherein said polynucleotide has the sequence shown in SEQ ID NO:l through 24.
14. The process of claim 11 wherein the bases of said polynucleotide are linked by pseudophosphate bonds that are resistant to cleavage by exonuclease or endonuclease enzymes.
15. The process of claim 14 wherein said bonds are phosphorothioate bonds.
16. The process of claim 11 wherein said administering is to the local site of said proliferation.
17. A process for treating a disease state involving the proliferation of vascular smooth muscle cells that comprises administering to a host mammal in need of such treatment an effective amount of a polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the gene for fibroblast growth factor receptor.
18. The process of claim 17 wherein said disease state is selected from the group consisting of vascular stenosis, post-angioplasty restenosis (including coronary, carotid and peripheral stenosis) , other non-angioplasty reopening procedures such as atherectomy and laser procedures, atherosclerosis, atrial-venous shunt failure, cardiac hypertrophy, vascular surgery, and organ transplant.
19. The process of claim 17 wherein said polynucleotide has the sequence shown in SEQ ID NO:l through 24.
20. The process of claim 17 wherein the bases of said polynucleotide are linked by pseudophosphate bonds that are resistant to cleavage by exonuclease or endonuclease enzymes.
21. A polynucleotide of about 10 to about 50 nucleic acid bases in length, said polynucleotide hybridizing to the about 5 to about 25 nucleic acid bases flanking the start codon of the mRNA for fibroblast growth factor.
22. The polynucleotide of claim 21 wherein said gene for fibroblast growth factor receptor is human fibroblast growth factor receptor.
23. The polynucleotide of claim 21 that is an antisense molecule having the sequence shown in SEQ ID
NO:l through 22.
24. The polynucleotide of claim 21 wherein the bases of said polynucleotide are linked by pseudophosphate bonds that are resistant to cleavage by exonuclease or endonuclease enzymes.
EP94907109A 1992-12-31 1993-12-28 Antisense molecules directed against a fibroflast growth factor receptor gene family Withdrawn EP0677055A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US99970692A 1992-12-31 1992-12-31
US999706 1992-12-31
PCT/US1993/012600 WO1994015945A1 (en) 1992-12-31 1993-12-28 Antisense molecules directed against a fibroflast growth factor receptor gene family

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AU (1) AU6080194A (en)
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Publication number Priority date Publication date Assignee Title
US5783683A (en) * 1995-01-10 1998-07-21 Genta Inc. Antisense oligonucleotides which reduce expression of the FGFRI gene
AU3617097A (en) * 1996-07-26 1998-02-20 Michel Chretien Pro-protein converting enzyme
CA2203745A1 (en) 1996-07-26 1998-01-26 Robert Day Pro-protein converting enzyme
NZ584827A (en) * 2007-10-01 2012-10-26 Isis Pharmaceuticals Inc Antisense modulation of fibroblast growth factor receptor 4 expression
CA2839437A1 (en) 2011-06-16 2012-12-20 Isis Pharmaceuticals, Inc. Antisense modulation of fibroblast growth factor receptor 4 expression

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Publication number Priority date Publication date Assignee Title
EP0529076B1 (en) * 1990-04-27 1995-12-06 Takeda Chemical Industries, Ltd. Proteins with fibroblast growth factor receptor activity
US5135917A (en) * 1990-07-12 1992-08-04 Nova Pharmaceutical Corporation Interleukin receptor expression inhibiting antisense oligonucleotides

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9415945A1 *

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AU6080194A (en) 1994-08-15
CA2152903A1 (en) 1994-07-21

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